Many therapeutically relevant recombinant proteins are produced in a variety of host organisms. Most proteins can be expressed in their native form in eukaryotic hosts such as CHO cells. Animal cell culture generally requires prolonged growing times to achieve maximum cell density and ultimately achieves lower cell density than prokaryotic cell cultures (Cleland, J. (1993) ACS Symposium Series 526, Protein Folding: In Vivo and In Vitro, American Chemical Society). Additionally, animal cell cultures often require expensive media containing growth components that may interfere with the recovery of the desired protein. Bacterial host expression systems provide a cost-effective alternative to the manufacturing scale production of recombinant proteins. Numerous U.S. patents on general bacterial expression of recombinant proteins exist, including U.S. Pat. Nos. 4,565,785; 4,673,641; 4,795,706; and 4,710,473. A major advantage of the production method is the ability to easily isolate the product from the cellular components by centrifugation or microfiltration. See, e.g., Kipriyanov and Little, (1999) Molecular Biotechnology, 12: 173-201; and, Skerra and Pluckthun, (1988) Science, 240: 1038-1040.
However, bacterial expression systems such as E. coli lack the cellular machinery to facilitate proper refolding of the proteins and generally do not result in the secretion of large proteins into the culture media. Recombinant proteins expressed in bacterial host cells are often found as inclusion bodies consisting of dense masses of partially folded and misfolded reduced protein. See, e.g., Baneyx, (1999) Current Opin. Biotechnology 10:411-421; and, Villaverde and Carrio, (2003) Biotech. Letts. 25:1385-1395. Proteins may also be expressed without forming inclusion bodies. See, e.g., Id. Typically in inclusion bodies, the recombinant protein is generally inactive.
Additionally, refolding often produces misfolded and disulfide-linked dimers, trimers, and multimers. (Morris et al., (1990) Biochem. J., 268:803-806; Toren et al., (1988) Anal. Biochem., 169:287-299). This association phenomenon is very common during protein refolding, particularly at higher protein concentrations, and appears often to involve association through hydrophobic interaction of partially folded intermediates (Cleland and Wang, (1990) Biochemistry, 29:11072-11078).
Misfolding occurs either in the cell during fermentation or during the isolation procedure. Proteins recovered from periplasmic or intracellular space must be solubilized and the soluble protein refolded into the native state. See, e.g., Rudolph, Renaturation of Recombinant, Disulfide-Bonded Proteins From “Inclusion Bodies” in Modern Methods in Protein- and Nucleic Acid Research (Walter de Gruyter New York, 1990) pp. 149-172. In vitro methods for refolding the proteins into the correct, biologically active conformation are essential for obtaining functional proteins. Typical downstream processing of proteins recovered from inclusion bodies includes the dissolution of the inclusion body at high concentration of a denaturant such as urea followed by dilution of the denaturant to permit refolding to occur (see, U.S. Pat. Nos. 4,512,922; 4,511,502; and 4,511,503). See also, e.g., Rudolph and Lilie, (1996) FASEB J. 10:49-56; Fischer et al., (1993), Biotechnology and Bioengineering, 41:3-13; Misawa & Kumagai, (1999) Biopolymers 51:297-307; and, Clark, (1998) Current Opinion in Biotechnology, 9:157-163; and, Tsumoto et al., (2003) Protein Expression and Purification 28:1-8. Such recovery methods are regarded as being universally applicable, with minor modifications, to the recovery of biologically active recombinant proteins from inclusion bodies. These methods have been applied to heparin binding protein (HBP) such as VEGF (Siemeister et al. (1996) supra). These methods seek to eliminate random disulfide bonding prior to coaxing the recombinant protein into its biologically active conformation through its other stabilizing forces and may not eliminate improperly folded intermediates, provide homogenous populations of properly folded product, or provide sufficient amounts of the properly folded product.
Reversed micelles or ion exchange chromatography have been used to assist refolding of denatured proteins by enclosing a single protein within micelles or isolating them on a resin and then removing the denaturant (Hagen et al., (1990) Biotechnol. Bioeng. 35:966-975; Creighton (1985) in Protein Structure Folding and Design (Oxender, D. L. Ed.) pp. 249-251, New York: Alan R. Liss, Inc.). These methods have been useful in preventing protein aggregation and facilitating proper refolding. To alter the rate or extent of refolding, conformation-specific refolding has been performed with ligands and antibodies to the native structure of the protein (Cleland and Wang, (1993), in Biotechnology, (Rehm H.-J., and Reed G. Eds.) pp 528-555, New York, VCH). For example, creatine kinase was refolded in the presence of antibodies to the native structure (Morris et al., (1987) Biochem. J. 248:53-57). In addition to antibodies, ligands and cofactors have been used to enhance refolding. These molecules would be more likely to interact with the folding protein after formation of the native protein. Therefore, the folding equilibrium could be “driven” to the native state. For example, the rate of refolding of ferricytochrome c was enhanced by the extrinsic ligand for the axial position of the heme iron (Brems and Stellwagon, (1983) J. Biol. Chem. 258:3655-3661). Chaperone proteins and folding catalysts have also been used to assist with protein folding. See, e.g., Baneyx, (1999) Current Opinion in Biotechnology, 10:411-421; & Carrio & Villaverde, (2003) FEBS Letters 537:215-221. However, these methods are not always efficient or sufficient to produce quantities of protein product.
There is a need for new and more effective methods of folding and/or recovering recombinant proteins from a host cell culture, e.g., for the efficient and economical production of recombinant proteins in bacterial cell culture. These new and more effective methods provide for improved recovery of a highly purified biologically active properly refolded protein and that are generally applicable to manufacturing scale production of the proteins. The invention addresses these and other needs, as will be apparent upon review of the following disclosure.